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| Status |
Public on Jul 24, 2010 |
| Title |
Differential gene expression in Roseburia inulinivorans grown on inulin and starch |
| Platform organism |
Roseburia inulinivorans |
| Sample organism |
Roseburia inulinivorans DSM 16841 |
| Experiment type |
Expression profiling by array
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| Summary |
Roseburia inulinivorans is a recently identified motile representative of the Firmicutes that contributes to butyrate formation from a variety of dietary polysaccharide substrates in the human large intestine. Microarray analysis was used here to investigate substrate-driven gene expression changes in R. inulinivorans A2-194. A cluster of fructo-oligosaccharide (FOS)/inulin utilisation genes induced during growth on inulin included one encoding a b-fructofuranosidase protein that was prominent in the proteome of inulin-grown cells. This cluster also included a 6-phosphofructokinase and an ABC transport system, while a distinct inulin-induced 1-phosphofructokinase was linked to a fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS II transport enzyme). Real-time PCR analysis demonstrated that the b-fructofuranosidase and adjacent ABC transport protein showed greatest induction during growth on inulin, whereas the 1-phosphofructokinase enzyme and linked PTS II transport system were most strongly up-regulated during growth on fructose, indicating that these two clusters play distinct roles in the utilization of inulin. The R. inulinivorans B-fructofuranosidase was over-expressed in E. coli and shown to hydrolyse fructans ranging from inulin down to sucrose, with greatest activity on fructo-oligosacharides. Genes induced on starch included the major extra-cellular a-amylase and two distinct a-glucanotransferases together with a gene encoding a flagellin protein. The latter response may be concerned with improving bacterial access to insoluble starch particles.
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| Overall design |
RNA was purified from mid-exponential phase (OD650 = 0.4) cultures of R. inulinivorans grown on basal YCFA supplemented with a single substrate of either starch or inulin using the RNeasy RNA purification kit (Qiagen), and the mRNA component enriched using the MICROBExpress system (Ambion). The purified RNA (1 ug) was labelled by reverse transcription (Amersham), employing random nonamer extension incorporating either dCTP-Cy3 or dCTP-Cy5 dyes. In order to ensure reproducibility, and to obtain statistically significant results, the dye labelling was swapped for a second hybridisation. RNA purified from a separate biological replicate was labelled and hybridised twice in the same way.
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| Contributor(s) |
Scott KP, Martin JC, Chassard C, Clerget M, Potrykus J, Campbell G, Mayer C, Young P, Rucklidge G, Ramsay AG, Flint HJ |
| Citation(s) |
20679207 |
| |
| Submission date |
Jun 09, 2010 |
| Last update date |
Mar 22, 2012 |
| Contact name |
Gill Campbell |
| E-mail(s) |
G.Campbell@abdn.ac.uk
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| Phone |
+44 (0) 1224 716627
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| Fax |
+44 (0) 1224 716647
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| URL |
http://www.rowett.ac.uk
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| Organization name |
University of Aberdeen
|
| Department |
Rowett Institute of Nutrition and Health
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| Street address |
Greenburn Road
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| City |
Aberdeen |
| ZIP/Postal code |
AB21 9SB |
| Country |
United Kingdom |
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| Platforms (1) |
| GPL10020 |
Roseburia inulinivorans 5K array |
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| Samples (4)
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| GSM553706 |
Roseburia Inulinivorans_inulin3_starch5_A |
| GSM553707 |
Roseburia Inulinivorans_starch3_inulin5_A |
| GSM553708 |
Roseburia Inulinivorans_starch3_inulin5_B |
| GSM553709 |
Roseburia Inulinivorans_inulin3_starch5_B |
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| Relations |
| BioProject |
PRJNA128757 |
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